Mitochondria are dynamic organelles that house the machinery for ATP synthesis, fuel oxidation, synthesis of building blocks for cell growth, ion homeostasis, cell death, and even innate immunity. Defects in these organelles underlie a number of human diseases, ranging from devastating infantile syndromes to virtually all age-associated disorders such as cancer, neurodegeneration, and diabetes. The long-term goal of our laboratory is to achieve a systems-level understanding of this organelle that can serve as a foundation for studying mitochondria in health and in disease. This RO1 represents the founding grant in our laboratory and in the previous funding period enabled us to achieve an important first step towards this long-term goal: a near-complete protein parts list that we call MitoCarta. This high quality and accurate inventory consists of ~1100 genes encoding the mammalian mitochondrial proteome. This inventory is used widely by the biomedical research community and has fueled many important basic and clinical discoveries. Moving forward, what is very much needed is a systematic, functional annotation of MitoCarta. We have recently assembled a near-complete and validated lentiviral RNAi collection spanning the MitoCarta gene set. In this renewal proposal we aim to use this organelle-wide RNAi collection in combination with cell-based assays to Aim 1: Link the mitochondrial proteome to cell growth and viability, Aim 2: Link the mitochondrial proteome to physiology, and Aim 3: Link the mitochondrial proteome to the organelle's abundance and morphology. Through these three aims, we will systematically annotate the function of MitoCarta proteins. The resulting phenotypic matrix can be clustered to group genes together on the basis of experimentally defined function, or alternatively, to cluster experimental readouts to reveal new relationships amongst phenotypic variables. The proposed project promises to create a community resource that will be extremely useful and thereby fuel basic and clinical investigations of mitochondria. The proposal builds naturally on the momentum we have already generated during the last period and will help to establish mitochondria as a model for human systems biology.